The present invention relates to PEG-LHRH analog conjugates wherein the PEG unit is covalently bound to Ser4 either directly or via a bifunctional linker molecule, such as an amino acid. The process for their production as well as their use in the therapy, prognosis or diagnosis of the diseases, in which LHRH analogs"" administration is advisable, are further objects of the present invention.
Covalent attachment of the hydrophilic polymer polyethylene glycol, (PEG), also known as polyethylene oxide, (PEO), to molecules has important applications in biotechnology and medicine. In its most common form, PEG is a linear polymer having hydroxyl groups at each terminus:
HOxe2x80x94CH2xe2x80x94CH2O(CH2CH2O)nCH2CH2xe2x80x94OH
This formula can be represented in brief as HOxe2x80x94PEGxe2x80x94OH, where it is meant that xe2x80x94PEGxe2x80x94 represents the polymer backbone without the terminal groups:
xe2x80x9cxe2x80x94PEGxe2x80x94xe2x80x9d means xe2x80x9cxe2x80x94CH2CH2O (CH2CH2O)n CH2CH2xe2x80x94xe2x80x9d
PEG is commonly used as methoxy-PEGxe2x80x94OH, (m-PEG), in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to chemical modification.
CH3Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH
Branched PEGs are also in common use. The branched PEGs can be represented as R(xe2x80x94PEGxe2x80x94OH)m in which R represents a central core moiety such as pentaerythritol or glycerol, and m represents the number of branching arms. The number of branching arms (m) can range from three to a hundred or more. The hydroxyl groups are subject to chemical modification.
Another branched form, such as that described in PCT patent application WO 96/21469, has a single terminus that is subject to chemical modification. This type of PEG can be represented as (CH3Oxe2x80x94PEGxe2x80x94)pRxe2x80x94X, whereby p equals 2 or 3, R represents a central core such as lysine or glycerol, and X represents a functional group such as carboxyl that is subject to chemical activation. Yet another branched form, the xe2x80x9cpendant PEGxe2x80x9d, has reactive groups, such as carboxyl, along the PEG backbone rather than at the end of PEG chains.
In addition to these forms of PEG, the polymer can also be prepared with weak or degradable linkages in the backbone. For example, Harris has shown in U.S. patent application Ser. No. 06/026,716 that PEG can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis. This hydrolysis results in cleavage of the polymer into fragments of lower molecular weight, according to the above reaction scheme:
xe2x80x94PEGxe2x80x94CO2xe2x80x94PEGxe2x80x94+H2Oxe2x86x92xe2x80x94PEGxe2x80x94CO2H+HOxe2x80x94PEGxe2x80x94
As used herein, the term polyethylene glycol or PEG is meant to include all the above described derivatives.
The copolymers of ethylene oxide and propylene oxide are closely related to PEG in their chemistry, and they can be used instead of PEG in many of its applications. They have the following general formula:
HOxe2x80x94CH2CHRO(CH2CHRO)nCH2CHRxe2x80x94OH
wherein R is H or CH3.
PEG is a useful polymer having the property of high water solubility as well as high solubility in many organic solvents. PEG is also non-toxic and non-immunogenic. When PEG is chemically attached (PEGylation) to a water insoluble compound, the resulting conjugate generally is water soluble as well as soluble in many organic solvents.
Luteinizing hormone releasing hormone (LHRH or GnRH) is a decapeptide secreted by the hypothalamus and capable of inducing the release of both LH and FSH. It has the following formula: pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO: 1).
LHRH can either stimulate pituitary gonadotropin secretion or be a potent inhibitor. When administered in a precise pulsatile pattern, LHRH can restore the normal cyclic gonadotropin secretion. Pulsatile administration of LHRH using a computerized pump was used with good results in the induction of ovulation in anovulatory women with hypothalamic dysfunction. When administered chronically, LHRH or its agonists proved to be potent inhibitors of gonadotropic secretion, providing a temporary (fully reversible) gonadotropin specific medical hypophisectomy.
To date, thousands of LHRH analogs have been synthesized that can act either as agonists or antagonists. In order to produce LHRH antagonists, which work by receptor occupancy, it is necessary to substitute several amino acids on the LHRH molecule. Antagonists also require precise topological features to achieve high binding affinity to the receptor. There are many recently synthesized LHRH analogs in which the amino acids contain aromatic or other functional groups capable of the so-called hydrotropic interaction. The use of LHRH antagonists with their immediate inhibition of gonadotrophin release may be useful in therapeutic areas, such as contraception and in treatment of hormone-dependent disorders. In the case of hormone-dependent tumors, avoiding the initial stimulatory phase produced by LHRH agonists may be a particular advantage. For a review on LHRH analogs, see Karten and Rivier, 1986.
Antide, in particular, is a potent LHRH antagonist, with formula, biological activity and preparation as described in EP Patent 377,665 and reported here below. 
[Acetyl-D-3-(2xe2x80x2-Naphthyl)-alanine)1, D-(4-Chlorophenyl)-alanine2, D-3-(3xe2x80x2-Pyridyl)-alanine3, Lysine (NE-Nicotinoyl)5, D-Lysine(NE-Nicotinoyl)6, Lysine(NE-Isopropyl)8, D-Alanine10]-Gonadotropin Releasing Hormone (GnRH).
From studies carried out by the present inventors, it was found, for example, that antide has a very poor solubility in 0.9% NaCl solution (solubility 25 xcexcg/ml) or other isotonic media such as phosphate buffered saline (solubility was 16 xcexcg/ml). Previous formulations of antide (e.g., antide 1 mg/ml in 5% glucose) have shown poor bioavailability and pharmacokinetic reproducibility.
Covalent attachment of PEG to peptides is a potentially useful approach for delivering water insoluble peptide drugs as shown by Felix (A. M. Felix in J. M. Harris and S. Zalipsky, Eds., Poly(ethylene glycol) Chemistry and Biological Applications, A.C.S Symposium Series 680, pp 218-238, A.C.S. Washington, D.C., 1997).
JP patent application JP 3148298 describes peptides-(e.g., including GnRH) PEG conjugates obtained by reacting the guanidino group, present for example in the arginine residue, with PEG, while protecting the amino groups present in the molecules.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicants at the time of filing and does not constitute an admission as to the correctness of such a statement.
This invention provides novel PEG-LHRH analogs conjugates wherein a PEG unit is covalently bound to Ser9 either directly or via a bifunctional linker molecule, such as an amino acid. PEG or PEG-linker molecule is bonded, specifically, to the alcohol function of the serine residue. The linkage between the LHRH analog and the polyethylene glycol or the PEG-linker molecule in these conjugates is subject to hydrolysis at hysiological pH (7.2-7.4) and is preferably also subject to hydrolysis by esterases present in the blood.